Numerous methods have been developed for the immobilization of proteins and other biomolecules onto solid or liquid supports. A description of these methods can be found in general reviews such as that given by Mosbach, 1976, Methods in Enzymology, Vol. 44; Weetall, 1975, Immobilized Enzymes, Antigens, Antibodies, and Peptides; or Kennedy et al., 1983, Solid Phase Biochemistry, Analytical and Synthetic Aspects, Scouten, ed., pp. 253-391. The most commonly used methods are adsorption or covalent binding to the support.
Adsorption is the oldest and simplest method for protein immobilization. To effect immobilization, a solution of the protein is contacted with a support material such as alumina, carbon, an ion-exchange resin, cellulose, glass or a ceramic. Although the immobilization procedure may be simple, the interactions involved in the adsorption process are complex and include charge-charge, van der Waals and hydrophobic interactions, and hydrogen bonding. The adsorption method has the advantages of low cost, extreme simplicity, mild immobilization conditions and the ability to regenerate the support. The main limitation of this method is the relatively weak interaction between the protein and the support, which may result in desorption of the protein upon changes in pH and ionic strength. The often undefined nature of these interactions also can limit their use.
The most frequently used immobilization technique is the covalent binding of the protein to chemically activated solid supports such as glass, synthetic polymers, and cross-linked polysaccharides. (Generally, this technique results in a protein which is immobilized in a more stable fashion than protein immobilized by adsorption.) An example of this method is the cyanogen bromide activation of polysaccharide supports, e.g., agarose.
Although these traditional supports have been used in many applications, they suffer from some limitations. The polysaccharide supports are compressible, which limits their application in column configurations at high flow rates. These supports are also susceptible to microbial attack. Silica supports are not stable under alkaline conditions. Polymeric supports are also not chemically inert, and usually have a specific gravity close to 1, which results in long settling times in batch operations. Moreover, all of these supports exhibit varying degrees of nonspecific binding of unwanted proteins. The use of solid and liquid fluorocarbon supports overcome many of these limitations. Fluorocarbons are chemically inert and mechanically stable. The high specific gravity of fluorocarbon supports results in rapid settling in batch operations. However, it is difficult to activate fluorocarbon supports for immobilization.
Fluorocarbon polymers have been used as supports to which biomolecules have been attached by adsorption [Fishman U.S. Pat. No. 3,843,443, issued on Oct. 22, 1974; WO 8603-840-A filed by Rijskuniv Groningen; Danielson and Siergiej, Biotechnol. Bioeng. 23, 1913-1917 (1981); Siergeiej, Dissertation Abstracts, Int. B., Volume 44, 153 (1983)]. Because these methods rely on simple adsorption of the biomolecule onto the support, the attachment is relatively weak. Consequently, some or all of the immobilized biomolecule is lost during use. In addition, a significant loss of biological activity of the biomolecule results upon adsorption.
Busby et al. (U.S. Pat. No. 4,317,879, issued Mar. 2, 1982) disclose the covalent attachment of the enzyme glucose oxidase to a fluorocarbon membrane. The membrane was first etched with a sodium dispersion in naphthalene, followed by paraformaldehyde linking of the enzyme. This method requires severe chemical conditions to activate the fluorocarbon surface for covalent binding to the enzyme.
Hato et al., (U.S. Pat. No. 4,619,897, issued Oct. 23, 1986) disclose the immobilization of enzymes onto a fluorine resin membrane which is made hydrophilic on one side by penetration of a perfluoroalkyl surface active agent to a prescribed depth. The asymmetrically functional membrane obtained is then treated with an enzyme and a cross-linking agent such as glutaraldehyde to effect immobilization. In this approach, the fluorocarbon surface is not activated for covalent attachment of the enzyme. Rather, the enzyme is crosslinked within the pores of the wetted membrane. This approach is limited to porous fluorocarbon membranes.
The use of perfluorocarbon polymer-based supports for enzyme immobilization and affinity chromatography is described in U.S. Pat. No. 4,885,250 issued Dec. 5, 1989. In this method the biomolecule is first modified by reaction with a perfluoroalkylating agent such as perfluorooctylpropylisocyanate described in copending application Ser. No. 134,028, now U.S. Pat. No. 4,954,444. Then, the modified protein is adsorbed onto the fluorocarbon support to effect immobilization. This procedure works well for the immobilization of many biomolecules, particularly immunoglobulins. However, substantial loss of biological activity results for some proteins because of the need to use organic solvents (16% v/v) in the perfluoroalkylation reaction, the hydrophobic nature of the fluorocarbon support, and the need for multipoint modification of protein to obtain secure immobilization. Multipoint modification of the biomolecule is required because of the mono(fluoroalkyl) reagents used. In addition, the mono(fluoroalkyl) reagents desorb from the support in the presence of high levels of organic solvents, e.g., about 50% or greater.
A revised method for immobilization onto fluorocarbon surfaces or non-fluorocarbons which contain a fluorocarbon interlayer attached to the surface of the core has been described in copending application Ser. No. 413,867, now U.S. Pat. No. 5,079,155. In this revised method, a perfluoroalkylating reagent containing a mono(fluoroalkyl) anchor group, a hydrophilic spacer arm, and a reactive group for covalent coupling to the protein is adsorbed onto the surface of the support. An aqueous solution of the protein is added to effect immobilization. This approach overcomes many of the limitations of the original method. Higher retention of activity, particularly for enzymes, is obtained because totally aqueous solutions of the enzyme are used, and the surface of the support is more hydrophilic. This approach also provides a preactivated support which is more convenient to use. However, the support with the adsorbed reagent is not stable because the reagent is secured by only one fluoroalkyl anchor group. In addition, multipoint attachment of the biomolecule is required for secure immobilization.
In copending application Ser. No. 413,867 a method for forming solid supports for size exclusion and ion-exchange separations is described. The support is prepared by forming a fluorocarbon interlayer by attaching a fluorocarbon to the surface of a solid carrier, followed by treatment with a fluorosurfactant. The use of a neutral fluorosurfactant results in a size exclusion support, while a charged fluorosurfactant provides an ion-exchange support. The stability of these supports is limited because of the mono(fluoroalkyl) anchor group which adsorbs to the fluorocarbon surface.
Giaver, (U.S. Pat. No. 4,619,904, issued Oct. 28, 1986) describes the use of fluorocarbon emulsions in agglutination immunoassays. The emulsions were formed by adding a fluorinated polar molecule such as pentafluorobenzoyl chloride to a fluorocarbon liquid. The resulting emulsion was contacted with an aqueous solution of the protein. Again, mono(fluoroalkyl) anchor groups were used to immobilize the protein.
Lowe et al. in copending application Ser. No. 428,154, describe the attachment of biomolecules to fluorocarbon surfaces by means of a polymer such as poly(vinyl alcohol), which has been chemically modified to contain a significant number of perfluoroalkyl groups. Although this approach provides multiple fluoroalkyl anchor groups for secure attachment to the fluorocarbon surface, the number of anchor groups is difficult to control and reproduce.
De Miguel et al., Chromatographia, Vol. 24, 849-853, 1987, describe the strong retention of phenyl-D-glucopyranoside, modified with multiple fluorocarbon chains, on fluorocarbon bonded phases under reversed phase conditions. The authors speculate that such strong retention may allow dynamic anchoring of biomolecules. No examples were provided. The compounds described cannot be used for immobilization because they contain no reactive group to couple to the biomolecule. The major difference between the phenyl-D-glucopyranosides of De Miguel et al., and the present invention is that their compounds do not contain a spacer arm and reactive group for covalent binding to the biomolecule.
One object of this invention is to provide a superior composition for modifying fluorocarbon surfaces that permit good retention of biomolecules.
Another object of this invention is to provide a reagent for facilitating immobilization of biomolecules on a support with retention of biological activity.
Yet another object of this invention is to provide a method for forming a solid support using the superior reagents described herein.
Still another object of this invention is to provide a process for immobilizing a biomolecule onto a fluorocarbon surface.